Immunolocalisation within neural tissue of MADM - A novel metalloprotease with a potential integrin-binding domain.
thesisposted on 19.11.2015, 08:50 by Sally A. Mitchell
A metalloprotease previously isolated from bovine brain was shown to be a member of a family which includes both mammalian proteins and snake venom metalloproteases containing a potential integrin-binding (disintegrin) domain. The bovine enzyme was named MADM for Mammalian-Disintegrin Metalloprotease (Howard et al., 1996; Biochemical Journal 371; 45-50). The present studies were undertaken to generate antibodies reactive with the rat MADM homologue and to use these for immunolocalisation of the protein in rat central nervous system (CNS). A truncated rat MADM homologue was initially isolated from a commercial brain cDNA library, and the sequence encoding the mature protein was completed using the technique of 5'-RACE (rapid amplification of cDNA ends). It was found that the rat MADM sequence had a very high degree of identity at both the nucleic acid and the amino acid level with bovine MADM, possibly implying a critical function for this protein. An antigenicity plot of the mature rat protein was used to design peptides for anti-MADM antibody production. Although the level of immunogenicity was generally low, two specific polyclonal antisera were produced in rabbits, and used in Western blotting experiments to demonstrate the presence of MADM protein in a range of rat tissues and cultured cell lines. Within adult rat CNS, the MADM protein was localised by immunohistochemistry to microglial cells of the white matter. The patterns of expression of 1, 2, 3, and 4-integrin subunits within the CNS were also examined, with the intention of identifying potential candidates for MADM's cognate integrin; only 2 integrins have been shown to co-localise with MADM to microglia. Ramified microglia of normal adult rat brain were shown to express low levels of MADM, and the protease was not demonstrated to be upregulated by reactive microglia either during early postnatal development or in the vicinity of a cerebral stab wound. Fluorescent confocal microscopy used to examine MADM expression by isolated microglia demonstrated that in vitro, MADM was found intracellularly, and could not be induced to move to a cell surface location even on microglia activated by cytokines; this intracellular localisation is in direct contradiction of the structural evidence pointing to a transmembrane position for MADM. However, MADM was found to be expressed on the surface of cultured mouse NSO myeloma cells, a result which may reflect a facet of the immortalisation process, or may imply that MADM is dependent on specific integrin expression for its surface localisation.